Use of poxviruses as enhancer of specific immunity

ABSTRACT

The invention relates to a method for enhancing the specific immune response against an immunogenic compound which comprises administering the immunogenic compound together with a poxvirus recombinant and a vaccinal antigen, which is not a poxvirus. The immunological material may be any biological material useful as a vaccine e.g., a polypeptide characteristic of a pathogenic microorganism or associated with a tumoral disorder, a DNA plasmid encoding a peptide or a polypeptide characteristic of a pathogenic microorganism or a tumor-associated antigen, or an hapten coupled to a carrier molecule. The poxvirus may be a live, attenuated or inactivated virus or a recombinant virus. Recombinant virus may encode a heterologous polypeptide such as chemokines, cytokines or co-immunostimulatory molecules or an homologous polypeptide, which is immunologically cross reactive with the immunogenic polypeptide or peptide.

The present invention relates to a method for enhancing the specificimmune response against an immunogenic compound, which comprisesadministering the immunogenic compound together with a poxvirus,recombinant or not.

Smallpox, a human infectious disease due to a vaccinia virus, wasdeclared eradicated from the globe in 1980. This unique success was madepossible by the availability of an effective virus-attenuated vaccine.Concurrent with the smallpox eradication and the cessation ofvaccination, a new use for the vaccinia virus was proposed (Panicali &Paoletti, PNAS (1982) 79:4927). Utilizing molecular cloning techniques,it became possible to express genes from foreign pathogens in vacciniavirus providing new approaches to vaccination.

Since then, the original technology has been applied to the wholepoxvirus family, including not only the vaccinia virus but alsoavipoxviruses such as fowlpox and canarypox. In order to address theissue of safety, a strategy was developed to genetically engineer ahighly attenuated vaccinia virus such as the Copenhagen strain thatwould still retain the ability to induce vigorous immunological responseagainst extrinsic antigens. A number of poxvirus constructions have beentested in clinical trials. As a matter of example, they includerecombinant vaccinia and canarypoxviruses expressing HumanImmunodeficiency Virus (HIV) or Plasmodium falciparum antigens. Further,it has already been proposed to combine, in an immunization protocol, aprime-administration using a recombinant poxvirus vector andbooster-administrations of the purified polypeptide as encoded by therecombinant vector (See e.g., Excler & Plotkin, AIDS (1997) 11 (suppl.A): S127). Such immunization protocols are commonly referred asprime-boost protocols and are very advantageous in a number of cases, inparticular for AIDS treatment or prevention.

Prime-boost protocols are however unpractical both for physicians,manufacturers and sellers, in that they require two differentpharmaceutical products that have to be each identified and licensed fortheir specific use (priming or boost).

It has now been found that poxvirus particles may be useful as enhancerof specific immunity. Indeed, it has been observed that the immuneresponse against a vaccinal antigen, such as an HIV or an influenzavirus protein, is enhanced, when it is mixed with a poxvirus,recombinant or not. Additionally, It has also been found that animmunization protocol exclusively using a composition comprising apolypeptide and a poxvirus encoding this polypeptide, may be just asgood as a prime-boost protocol. It has also surprisingly been found thatthe observed immunization effect is not a mere additional effect, butresults from a synergism effect between the two components.

Therefore, the invention provides for:

(i) The use of a poxvirus for the manufacture of a pharmaceuticalcomposition comprising an immunogenic compound for inducing an immuneresponse in a vertebrate, wherein the poxvirus is able to enhance aspecific immune response to the immunogenic compound.

(ii) The use of a mixture comprising (a) an immunogenic compound whichcomprises at least one antigenic determinant characteristic of apathogenic microorganism or is cross-reactive with a tumor-associatedantigen (TAA) and (b) a poxvirus; in the manufacture of a medicament tobe administered to a vertebrate for treating or preventing an infectioninduced by the pathogenic microorganism or a tumoral disordercharacterized by the malignant expression of the TAA; whereby saidpoxvirus enhances the specific immune response of the vertebrate againstsaid immunogenic compound.

(iii) A pharmaceutical composition comprising (a) an immunogeniccompound and (b) a poxvirus encoding an heterologous polypeptide whichis selected from the group consisting of adhesion molecules,co-immunostimulatory molecules, apoptotic factors, cytokines, chemokinesand growth hormones.

(iv) A pharmaceutical composition comprising (a) an immunogenic compoundwhich is a first polypeptide and (b) a poxvirus encoding an heterologouspolypeptide,which has an amino acid sequence identical to the amino acidsequence of the first polypeptide.

(v) A pharmaceutical composition comprising (a) an immunogenic compoundwhich is a DNA plasmid encoding a first polypeptide and (b) a poxvirusencoding a second heterologous polypeptide, which has an amino acidsequence identical to the amino acid sequence of the first polypeptide.

(vi) A method for enhancing the specific immune response of a vertebrateto an immunogenic compound, which comprises administering to thevertebrate the immunogenic compound together with a poxvirus, wherebythe poxvirus enhances the specific immune response to the immunogeniccompound.

(vii) A method for treating or preventing in a vertebrate, a disordereither induced by a pathogenic microorganism or characterized by themalignant expression of a T.A.A, which comprises administering to thevertebrate, (a) an immunogenic compound which comprises at least oneantigen determinant characteristic of the pathogenic microorganism or atumor-associated antigen together with (b) a poxvirus; whereby aspecific immune response to the immunogenic compound is induced in thevertebrate and whereby the poxvirus enhances the specific immuneresponse.

(viii) A method for enhancing the specific “in vitro” immunostimulationof cells from an immune system against a specific immunogenic compound,which comprises (a) recovering cells from a vertebrate, (b) “in vitro”incubating the cells with the immunogenic compound together with apoxvirus, whereby the cells are immunostimulated against the immunogeniccompound and whereby the poxvirus enhances the immunostimulation and (c)administering the immunostimulated cells obtained from step (b) to avertebrate.

In a general manner, there exist two types of immunity: the innateimmunity and the acquired immunity. The former which is phylogeneticallyolder brings into play soluble molecules, i.a. complement factors andcells, such as NK cells or macrophages, which are innately programmed todetect noxious substances produced by pathogenic microorganisms and toprovide for rapid but often incomplete antimicrobial host defense. Theinnate immune system intervenes as the first line of defense when aninfectious agent attacks an individual. On the other hand, the innateimmune system can not be educated by the antigens expressed by thepathogenic microorganisms or tumor cells during the life of anindividual and in this respect; the innate immunity is confounded withthe natural immunity. By contrast, the acquired immune system bringsinto play antigen-specific B and T lymphocyte clones the affinity ofwhich increases by the time consecutively to repeated contacts with thespecific antigen. Moreover, some of them behave as memory lymphocytes,since they have a long lasting life and are able to proliferate andexpand rapidly consecutively to a further contact with a specificantigen, so that these memory lymphocytes contribute to the long termprotection of an individual to infectious microorganisms. An essentialgoal of vaccination is to provide for these memory lymphocytes.

Accordingly, by “specific immune response” is meant a specific humoraland/or a specific cellular immune response against the immunogeniccompound of the pharmaceutical composition. In the present invention,the specific humoral immune response includes both systemic and mucosalantibody responses since, to feature the humoral response, one may referto all types of specific antibodies, i.e. IgM, all subclasses of IgG andIgA, that may be elicited by the pharmaceutical composition. Thespecific lymphoproliferative response and the specific cytotoxic Tlymphocyte (CTL) response preferentially are the main parameters of thespecific cellular immune response.

For use in the present invention, the immunogenic compound may be achemical or a biological material that is able to induce a humoral orcellular immune response in a vertebrate. A biological material may bee.g., an attenuated, inactivated or killed virus (to the exception of apoxvirus); a bacterial strain; a pseudovirion; a bacterial extract; acapsular polysaccharides; a peptide or a polypeptide foundtumor-associated, cross-reactive with a TAA or characteristic of apathogenic agent; or a DNA plasmid encoding a peptide or a polypeptideas described above. As an example of chemical material, a hapten coupledto a carrier protein is cited.

By “hapten” is meant a molecule, generally of low molecular weight,which is unable to trigger an antibody response by itself, but capable,after coupling with a carrier, to induce a specific antibody responsewhich interacts specifically with the hapten molecule. For use in thepresent invention, such an hapten may be a peptide which amino acidsequence is at least 5 to 6 amino acid long (minimal size of an epitope)but of low molecular weight, a chemical molecule (such asdinitrophenol), or a drug. In a particular embodiment of the presentinvention, a mixture according to the invention may be intended to treatdrug addiction and to this end, may comprise a poxvirus, mixed with adrug, such as cocaine, coupled to a carrier molecule to induce anantibody response against the drug, in order to hamper both its fixationon the target cells, tissues or organs and the triggering of itsnarcotic effects. Methods of coupling a hapten to a carrier molecule areof common use for a man skilled in the art.

By “polypeptide” or “protein” is meant any chain of amino acids,regardless of the length or post-translational modification (e.g.,glycosylation or phosphorylation). Both terms are used interchangeablyin the present application.

Advantageously, immunogenic polypeptides may be polypeptidescharacteristic of a pathogenic microorganism i.e. a virus, bacteria oran eucaryotic parasite, or tumor-associated antigens (that are mammalianor avian antigens which are not normally expressed; their malignantexpression is characteristic of a tumoral disorder) such as tyrosinase,the MAGE protein family, the CEA, the ras protein, mutated or not, thep53 protein, mutated or not, Muc1, CEA and pSA.

For use in the present invention, immunogenic polypeptides may haveamino acid sequences corresponding to the complete or partial sequenceof naturally occurring polypeptides. They may also have a sequencederived by amino acid deletion, addition or substitution from thenaturally occurring sequences as far as they behave as immunologicequivalents i.e., they are able to induce an immune response against thepathogenic microorganisms from which they derive or against the tumor.In other terms, an immunogenic polypeptide is also meant to include anypolypeptide that is immunologically cross-reactive with a naturallyoccurring polypeptide found in a pathogenic agent or tumor-associated.

By “immunologically cross-reactive polypeptides” is meant polypeptidesthat can be recognized by antibodies, e.g. polyclonal antibodies, raisedagainst each of the polypeptides used separately, and advantageously ina substantially purified form.

As a matter of example, the polypeptide may be an HIV antigen such asthe env, gag, pol or nef protein. An HIV antigen is also meant toinclude any polypeptide that is immunologically cross-reactive with anaturally occurring HIV protein. For example, an HIV env protein may bethe gp160 env precursor, or the gp120 or gp41 sub-unit. The gp160precursor may be a soluble, non-cleavable precursor obtained by mutationof the cleavage site and deletion of the transmembrane region asdescribed in U.S. Pat. No. 5,672,689. The precursor may also betruncated so that the C-terminal part of the gp41 region is removed(intracytoplasmic domain). The precursor may also be a hybrid precursor,combining in a single molecule, env sequences from various HIV strains.An HIV gag antigen may be the complete p55 precursor, the p13, p18 orp25 that naturally derive from p55, or any immunogenic gag proteinfragment. In fact, a large variety of polypeptides may be substitutedfor the naturally occurring HIV env, gag, pol or nef proteins, yetretaining their immunogenic properties.

As an additional example the polypeptide may be an influenza peptide orpolypeptide which comprises the virus envelope components such as thehaemagglutinin and the neuraminidase and the virus internal componentssuch as the protein M, the non-structural proteins and thenucleoprotein. An influenza peptide or polypeptide is also meant toinclude any precursor form of the mature envelope or internal proteinsthat are immunologically cross reactive with them. Likewise, thepolypeptide or peptide may be any kind of haemagglutin or neuraminidaseof the influenza virus since there are numerous antigenic variants ofthese two proteins.

For use in the present invention, the polypeptide characteristic of apathogenic agent that is physically present in the composition may bepurified from the pathogenic agent itself or recombinantly produced.Advantageously tumor-associated antigens (TAAs) as well will be producedby recombinant means. Standard expression vectors, promoters,terminators, etc and recombinant methods are now of common use for a manskilled in the art and recombinant expression can be readily achievedonce an appropriate DNA sequence corresponding to the polypeptide isavailable. In a particular embodiment, polypeptides may be recombinantlyproduced as fusion polypeptides (i.e., a polypeptide fused through itsN- or C-terminal end to any other polypeptide (hereinafter referred toas a peptide tail), using appropriate expression vectors, such as thepMal-c2 or pMal-p2 systems of New England Biolabs in which the peptidetail is a maltose binding protein, or the His-Tag system available fromNovagen.

An immunogenic compound, e.g., a polypeptide physically present in acomposition of the invention is advantageously present in asubstantially purified form, i.e., it is separated from the environmentin which it naturally occurs and/or is free of the majority of thepolypeptides that are present in the environment in which it wassynthesized.

As mentioned above, the immunogenic compound may also be a DNA plasmidunable to replicate in eucaryotic cells, comprising a DNA sequenceencoding a peptide or a polypeptide, this latter being defined as hereinabove, under the control of an appropriate promoter which allows thepeptide or polypeptide to be expressed in eucaryotic cells aftertransfection by the recombinant plasmid. As a matter of example, the CMV(Cytomegalovirus) early promoter is broadly used for the expression of aheterologous peptide or polypeptide in human cells transfected with DNAplasmid encoding peptide or polypeptide.

In a particular embodiment of the present invention, a DNA plasmidadvantageously encodes a peptide comprising one or several epitopescharacteristic of a viral, bacterial, parasitic, or tumor-associatedpolypeptide. As a matter of example, it is well known thattumor-associated antigens, such as Her-2 neu, are often poor immunogens,because they are essentially “self” antigens. To overcome the lack ofimmunogenicity, it is commonly proposed to use as an immunogeniccompound, instead of DNA encoding the whole polypeptide, a DNA encoding“subdominant” epitopes selected from the polypeptide. This strategy isalso applicable to infectious microorganisms, such as HIV, Mycobacteriumtuberculosis or Plasmodium falciparum for which the protective antigensare not yet defined. In a particular embodiment of the invention, aimedat the induction or the enhancement of a specific CTL response in avariety of Major Histocompatibility Complex (MHC) contexts, apharmaceutical composition comprising a poxvirus mixed together with aDNA plasmid encoding customized peptides, may be useful. A customizedpeptide comprises or mimics an epitope selected throughout the wholeamino acid sequence of an antigen of a pathogenic micro-organism or atumor, as containing putative anchor motifs needed for binding tovarious MHC class I molecules (such as in humans, HLA-A1, HLA-A2,HLA-B7, . . . ). The customized peptides encoded by the plasmid may alltogether preferably trigger a specific CTL response in the main MHCcontexts, of a given vertebrate.

For use in the present invention, the poxvirus may be any virusbelonging to the poxviridae family. Accordingly, useful poxvirusesinclude, capripoxvirus, suipoxvirus, molluscipoxvirus, yatapoxvirus,entomopoxvirus, orthopoxvirus and avipoxvirus; these two latter beingpreferred. A typical orthopoxvirus is a vaccinia virus. A suitablevaccinia virus may be e.g., the highly attenuated Copenhagen strain orthe NYVAC vector that is derived from the Copenhagen strain by precisedeletion of 18 open reading frames (ORFs) from the viral genome asdescribed in Tartaglia et al, Virology (1992) 188:217. A typicalavipoxvirus is a canarypoxvirus or a fowl poxvirus. A suitablecanarypoxvirus may be e.g., the ALVAC vector obtained as described inTartaglia et al (supra). A suitable fowlpox vector may be e.g., theTROVAC vector which is a plaque-cloned isolate derived from the FP-1vaccine strain licensed for vaccination of 1 day old chicken (sold byMerial, Lyon, France) and described in Taylor et al, Vaccine (1988) 6:497.

A poxvirus for use in the present invention may be a live, attenuated orinactivated virus. By “live virus” is meant a virus that is fullycapable to reproduce its natural infectious cycle into sensitive cells,comprising virus entry, uncoating, gene expression, DNA replication,virus assembly, maturation and release. In a particular embodiment, alive virus may be attenuated. Attenuated virus may be obtained, e.g., byselection-of spontaneous mutants after repeated infectious cycles intosensitive cells, by selective pressure or deletion of non-essentialgenes using molecular biology tools. Nevertheless, whatever the processof attenuation, the viruses that are issued remain able to reproducethemselves into sensitive cells even if sometimes the spectrum ofsensitive cells can decrease. As a matter of example, it may be usefulto delete the vaccinia virus genome from K3L or E3L genes to render itmore sensitive to the action of interferons and consequently to reduceits host restriction range (Beattie E and al., (1996) Virus Genes, 12,89-94). As a matter of example a suitable live virus for use in humansmay be a canarypoxvirus, since in human cells such a virus exhibits anabortive infectious cycle. Additionally a suitable attenuated virus foruse in humans may be a NYVAC vector. By “inactivated virus” is meant avirus that is no more capable to reproduce its entire infectious cycleinto sensitive cells as a result of either a mechanical, chemical orphysical treatment. As may be easily understood, inactivation isparticularly advantageous when a non-recombinant poxvirus is used.

For use in the present invention, a poxvirus may be recombinant or not.A non-recombinant poxvirus does not encode any heterologous polypeptide.On the other hand, a recombinant virus is typically a virus in thegenome of which is inserted one or several foreign genes (e.g. anheterologous coding sequence located in the genome under the control ofa viral promoter allowing at least a transient expression in thevirus-infected cells).

A useful recombinant poxvirus encodes a heterologous peptide orpolypeptide that may be of any kind. In one embodiment of the invention,the peptide or the polypeptide may be a cytokine, such as interleukin-2.(IL-2), interleukin-3 (IL-3) interleukin-12 (IL-12), interleukin-15(IL-15), interleukin-18 (IL-18) and granulocyte macrophage-colonystimulating factor (GM CSF); a chemokine, such as RANTES (Regulated onActivation Normal T-cell Expressed and Secreted) and MCP1 (MonocyteChemotactic protein 1); a co-immunostimulatory molecule, such as B7,CD40, CD40L and ICAMs (inter cellular adhesion molecules); an adhesionmolecule; an apoptotic factor, such as p53 and TNF (tumor necrosisfactor); or an hormone such as a growth hormone.

In another embodiment, the immunogenic compound for use in the presentinvention is a peptide or a polypeptide and the admixed poxvirus encodesa heterologous peptide or polypeptide that cross-reacts with theimmunogenic compound. Accordingly, the invention also features the useof a poxvirus for the manufacture of a pharmaceutical compositioncomprising a first polypeptide; wherein the poxvirus encodes a secondpolypeptide which immunologically cross reacts with the firstpolypeptide. The encoded polypeptide may be the same as the one presentin the composition. In other words, the encoded polypeptide has an aminoacid sequence identical to that of the polypeptide present in thecomposition. Alternatively, the poxvirus may encode an immunogenicpolypeptide that is similar to the polypeptide present in thecomposition, although slightly different at the amino acid sequencelevel. In a particular embodiment, the immunogenic polypeptide presentas such in the composition originates from a particular pathogenicstrain and the poxvirus vector accompanying the polypeptide encodes anallelic variant thereof i.e., the same polypeptide but from anotherstrain. As a result, the polypeptide physically present and the encodedpolypeptide may have amino acid sequences slightly different, being atleast 70, 80, 90% or more identical. A composition comprising the HIV MNgp120 together with a poxvirus encoding HIV LAI gp120 is cited as amatter of example. In another embodiment, the sequences of both thepolypeptide physically present and the encoded polypeptide may derivefrom each other by addition, deletion or substitution of one or severalamino acids, provided that these polypeptides are immunologicallycross-reactive. As a matter of example, it is cited a compositioncomprising:

(i) HIV gp160 and a poxvirus encoding HIV gp120;

(ii) HIV gp160 in a soluble and non-cleavable form and a poxvirusencoding wild-type gp160;

(iii) HIV gag p55 and a poxvirus encoding gag p18; or

(iv) HIV gp120 and a poxvirus encoding HIV gp120-p18 hybrid protein; or

(v) HIV gp120, HIV p18 and a poxvirus encoding HIV gp120-p18 hybridprotein.

As illustrated in section (v) hereinabove, a composition of theinvention may comprise not only one but also two or more polypeptidespresent as such. The poxvirus may also encode several immunogenicpolypeptides, at least one being immunologically cross-reactive with apolypeptide physically present in the composition; or the compositionmay contain several poxviruses. Advantageously, when severalpolypeptides are present as such, the compositions of the inventionfurther contain a poxvirus that operatively encodes polypeptides, eachof them being two-by-two cross-reactive with the polypeptides physicallypresent. Alternatively, the composition may contain several poxviruses,-each of them encoding a polypeptide cross-reactive with a polypeptidephysically present. As understood by a man skilled in the art, a largevariety of combinations are possible.

Recombinant pox vectors may be constructed using the basic two-steptechnique of Piccini et al, (1987) in “Meth. In Enzymology” Acad. Press,San Diego and widely used for any pox vector as described in U.S. Pat.Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587 and 5,179,993. First,the heterologous DNA sequence to be inserted into the poxvirus is placedunder the control of a suitable poxvirus promoter able to directexpression of the sequence in avian or mammalian cells. The expressioncassette is then introduced into an E. coli plasmid that contains a DNAregion homologous to a non-essential locus of the pox vector DNA. Theexpression cassette is positioned so that it is flanked on both ends bypoxvirus homologous DNA sequences. The resulting plasmid is thenamplified by growth within E. coli and isolated. Second, the isolatedplasmid containing the expression cassette to be inserted is transfectedinto a cell culture, e.g. chick embryo fibroblasts, along with thepoxvirus. Recombination between homologous poxvirus DNA present on theplasmid and the viral genome gives a recombinant poxvirus modified bythe presence, in a non-essential region of its genome, of the expressioncassette containing the heterologous DNA sequence.

For use in the present invention, poxviruses, irrespective of whetherthey are recombinant or not, may be propagated on mammalian cells suchas Vero cells, BHK21 cells and Chick Embryo Fibroblasts (CEF), asdescribed in e.g., Piccini et al, and Taylor et al (supra). Oncepropagated, the viral particles may be merely harvested and clarified bycentrifugation. They may also be purified further according to Joklicket al, Virology (1962) 18:9.

Compositions and/or methods of the invention are useful for boththerapeutic and prophylactic purposes. When the immunogenic compound ischaracteristic of a pathogenic microorganism or a T.A.A., the specificimmune response induced upon administration of the compositions orresulting from the methods of the invention, is advantageouslyprotective against the pathogenic microorganism or the tumoral disorder.As a matter of example, there is a need to improve the current influenzavaccine which is not optimally protective in old people. Suchpharmaceutical compositions or methods of the invention provide forimproved protection over the flu vaccine of the prior art as exemplifiedin example 6.

Compositions of the invention can be manufactured in a conventionalmanner. In particular, the compounds can be formulated with apharmaceutically acceptable diluent or carrier e.g., water or a salinesolution such as phosphate buffer saline. In general, a diluent orcarrier can be selected on the basis of the mode and route ofadministration, and standard pharmaceutical practice. Suitablepharmaceutical diluents or carriers as well as pharmaceuticalnecessities for their use in pharmaceutical formulations are describedin Remington's Pharmaceutical Sciences, a standard reference text inthis field.

A composition of the invention may be administered to any kind ofvertebrate, i.a. to mammals or birds, in particular to humans. To thisend, one can use any conventional route in use in the vaccine fielde.g., via parenteral routes such as the intravenous, intradermial,intramuscular and sub-cutaneous route or mucosal routes such as nasal ororal routes. Especially, for the immunotherapy of cancer it may beuseful to administer the pharmaceutical composition intratumorally orinto the neighbor lymph nodes.

Compositions comprising a DNA plasmid as immunogenic compound, mayadvantageously be administered into the epidermis using a special devicesuch as a gene gun or an equivalent device, or by intramuscular route.Taking into account that most of poxvirus are able to infect epidermiscells, it is worth noticing that the composition of the invention andadvantageously a composition comprising a DNA plasmid mixed with apoxvirus is suitable for an intradermal or trancutaneous immunization asdescribed by Glenn GM et al, (1998), J. Immunol. 161:3211-3214.

In a general manner, the administration can be achieved in a single doseor repeated at intervals, e.g. repeated twice or more, one or two monthsapart.

In compositions of the invention, the appropriate dosage of thepoxvirus, and the immunogenic compound depends on various parametersunderstood by skilled artisans such as the vector and the immunogeniccompound themselves, the route of administration, the general status ofthe vertebrate to be vaccinated (weight. age and the like), the type ofimmune response that is desired and the tumoral or infectious site. Anefficient amount of the compounds is such that upon administration, animmune response against the compounds will be induced. For guidance, itis however indicated that the infectious titer (amount of virus able toinfect 50% of a cell culture) per dose of the poxvirus may suitablyrange from 10³ to 10⁹, preferably from 10⁵ to 10⁸ CCID50 (Cell CultureInfectious Dose 50). The polypeptide(s) physically present in thecomposition may amount from 10 μg to 1 mg, advantageously from 25 to 500μg, preferably from 50 to 200 μg; most preferably, a single dosecontains about 50-100 μg of polypeptide(s). Whenever a DNA plasmid isthe immunogenic compound, a convenient dose of DNA plasmid administeredmay amount from several ng to a few mg depending on the size of theanimal giving the composition. In human beings the suitable dose of DNAplasmid per immunization may range from 20 μg to 2500 μg as mentioned byWang R et al (1998), Science, 282, 476-480.

All the documents cited throughout the specification are incorporated byreference.

The invention is further explained and illustrated in the examples byreference to the figures described as follows.

FIGS. 1a and 1 b refer to Example 1 and show mean gp160 MN/LAI ELISAantibody titers (log) in guinea-pigs immunized twice by intramuscularroute (on days 1 and 29) with vCP205 and/or gp160 MN/LAI 4 μg (1 a) or40 μg (1 b).

FIGS. 2a and 2 b refer to Example 1 and show mean V3 MN ELISA antibodytiters (log) in guinea-pigs immunized twice by intramuscular route (ondays 1 and 29) with vCP205 and/or gp160 MN/LAI 4 μg (2 a) or 40 μg (2b).

In FIGS. 1a and 2 a: ∘ corresponds to group #1 (D1 and D29 gp160); corresponds o group #3 (D1: vCP205 and D29: gp160); ∇ corresponds togroup #5 (D1 and D29: vCP205+gp160), and ▾ corresponds to group #7 (D1and D29: vCP205).

In FIGS. 1b and 2 b: ∘ corresponds to group #2 (d1 and D29: gp160); corresponds to group #4 (D1: vCP205 and D29: gp160); ∇ corresponds togroup #6 (D1 and D29: vCP205+gp160); and ▾ corresponds to group #7 (D1and D29: vCP205).

FIG. 3 refers to Example 2 and shows CPpp antibody titers (log/ml) inguinea-pigs inoculated twice intramuscularly with various doses ofvCP205.  corresponds to group #3 (10{circumflex over ( )}4.8 CCID50); ▾corresponds to group #5 (10{circumflex over ( )}5.8 CCID50); and ▪corresponds to group #8 (10{circumflex over ( )}6.1 CCID50).

FIGS. 4a and 4 b refer to Example 2 and show gp160 MN/LAI ELISA antibodytiters (log/ml) in guinea-pigs inoculated twice intramuscularly withvarious doses of vCP205 and/or gp160 MN/LAI.

In FIG. 4a,  corresponds to group #1 (40 μg of gp160), ▾ corresponds togroup #2 (80 μg of gp160); U corresponds to group #4 (10{circumflex over( )}4.8 CCID50 of vCP205+40 μg of gp160); ▴ corresponds to group #6(10{circumflex over ( )}5.8 CCID50 of vCP205+40 μg of gp160 mixedtogether); and ♦ corresponds to group #7 (10{circumflex over ( )}5.8CCID50 of vCP205+40 μg of gp160 injected separately).

In FIG. 4b, ∘ corresponds-to group #3 (10{circumflex over ( )}4.8 CCID50of vCP205), ∇ corresponds to group #5 (10{circumflex over ( )}5.8 CCID50of vCP205), □ corresponds to group #8 (10{circumflex over ( )}6.1 CCID50of vCP205), ▪ corresponds to group #4 (10{circumflex over ( )}4.8 CCID50of vCP205+40 μg of gp160), ▴ corresponds to group #6 (10{circumflex over( )}5.8 CCID50 of vCP205+40 μg of gp160 mixed together),♦ corresponds togroup #7 (10{circumflex over ( )}5.8 CCID50 of vCP205+40 μg of gp160injected separately).

FIGS. 5 to 8 refer to Example 3 and show the mean ELISA antibody titers(log/ml) in macaques immunized intramuscularly with 10^(6.5) CCID50vCP205 and/or 100 μg gp160 MN/LAI adjuvanted or not (FIG. 5 gp160 ELISAantibody; FIG. 6: V3 MN ELISA antibody; FIG. 7: p24 LAI ELISA antibody;FIG. 8: CPpp ELISA antibody). ♦ corresponds to group #1; ∇ correspondsto group #2; ∘ corresponds to group #3; and ▾ corresponds to group #4.(▪ is irrelevant).

FIG. 9 refers to Example 3 and shows the HIV MN seroneutralizingantibody titers (log) in macaques immunized five times intramuscularlywith 10^(6.5) CCID50 vCP205 and/or 100 μg gp160 MN/LAI adjuvanted or notat weeks 0 (square-dotted box), 16 (hatched box) and 26 dotted box).Schemes A to D correspond respectively to groups #1 to #4.

FIGS. 10a and 10 b refer to Example 4 and show ELISA CPpp antibody (1 a)and gp160 MN/LAI antibody (1 b) mean titers in guinea-pigs primedintramuscularly with a mixture of gp160 MN/LAI (5 μg) and differentdoses of crude or purified CPpp, then boosted with 5 μg of gp160 MN/LAI.∘ corresponds to group #1;  corresponds to group #2; ∇ corresponds togroup #3; ▾ corresponds to group #4; and □ corresponds to group #5.

FIGS. 11a and 11 b refer to Example 5 and show ELISA IgG CPpp antibody(2 a) and gp160 MN/LAI antibody (2 b) mean titers in guinea-pigs primedintramuscularly with a mixture of gp160 MN/LAI (5 μg) and differentfractions of ALVAC-Luc (vCP297), either inactivated or not, then boosted(week 4) with 5 μg of gp160 MN/LAI. ∘ corresponds to group #1; corresponds to group #2; ∇ corresponds to group #3; ▾ corresponds togroup #4; and □ corresponds to group #5.

FIGS. 12 and 13 refer to example 6 and show respectively the IgG1 andIgG2a ELISA antibody titers specific for A/Texas in each individual agedDBA/2 mice immunized twice with either 3 μg of A/Texas (group 1), 2×10⁷CCID50 of CPpp and 3 μg of A/Texas (group 2) or 2×10⁷ CCID50 of CPpp(group 3). ▪ and ♦ correspond to mice of group 1 respectively after oneand two immunizations. ▴ and □ correspond to mice of group 2respectively after one and two immunizations. ♦ and ⋄ correspond to miceof group 3 respectively after one and two immunizations.

FIG. 14 refers to example 6 and shows the survival curves of the 3immunized groups after a lethal challenge with A/Taïwan. Δ correspondsto group 1; □ corresponds to group 2; ♦ corresponds to group 3.

FIG. 15 refers to example 6 and shows the morbidity curves of the 3immunized groups after a lethal challenge with A/Taïwan. Δ correspondsto group 1; □ corresponds to group 2; ♦ corresponds to group 3.

EXAMPLE 1 Simultaneous Immunization With ALVAC-HIV (vCP205) and gp160MN/LAI in Guinea Pigs

1A—vCP205 Preparation

vCP205, an ALVAC pox vector capable of expressing HIV proteins, isdescribed in Example 14 of WO 95/27507. Briefly, it contains a firstheterologous sequence encoding the env gp120 MN+the transmembrane regionof LAI gp41, and a second sequence encoding LAI (gag+protease); thesesequences are inserted in the C3 locus and placed under the control ofpromoters H6 and I3L.

Clarified vCP205 was produced on chick embryo fibroblasts in DMEM—HamF12 medium without serum, harvested in lactoglutamate and clarified bycentrifulgation. The preparation used hereinafter has a mean titer of10^(8.5) CCID₅₀/ml on QT35 cells.

Purified vCP205 was produced as described above and further purifiedaccording to Jokick et at, (supra). The VCP205 preparation in phosphatebuffer saline (PBS) 20 mM pH 7.2 (in the absence of Mg⁺⁺ and Ca⁺⁺) asused hereinafter, has a mean titer of in 10⁸ ⁸ CCID₅₀/ml on QT35 cells.

1B—gp160 MN/LAI Preparation

A recombinant vaccinia virus vector, VVTG9150, is used for gp160production. VVTG9150 operatively encodes a hybrid, soluble HIV-1 gp160in which the gp120 moiety derives from HIV-1 MN and the gp41trans-membrane part comes from the LAI isolate. The DNA sequencescorresponding to these two compounds are fused together using anartificial SmaI restriction site, which modifies neither the gp120, northe gp41 amino acid sequence. The construction of the two partners isbriefly described as follows.

The sequence encoding the MN gp120 was amplified from cellsSupT1infected with HIV-MN, using the PCR technique with oligonucleotideswhich introduce a SphI and SmaI restriction sites respectively locatedimmediately downstream of the sequence encoding the leader peptide andupstream of the cleavage sites located between gp120 and gp41.

The sequence encoding the LAI gp41 was produced as follows: The completeHIV-1 LAI env coding sequence was placed under the control of thevaccinia pH5R promoter. Several modifications were introduced into thisencoding sequence. First a SphI restriction site was created immediatelydownstream of the sequence encoding the leader peptide, without alteringthe amino acid sequence. Second, a SmaI restriction site was createdimmediately upstream of the sequence encoding the cleavage sites betweengp120 and gp41, without altering the amino acid sequence. Third, the twocleavage sites in position 507-516 (amino acids numbered according toMyers et al, In: Human retroviruses and AIDS (1994) Los Alamos NationalLab. (USA)) were mutated (original sequence KRR . . . REKR mutated intoQNH . . . QEHN). Fourth, the sequence encoding the transmembranehydrophobic peptide IFIMIVGGLVGLRIVFAVLSIV (amino acids 689-710 in Myerset al (supra)) was deleted. Fifth, a stop codon was substituted for thesecond E codon of the sequence encoding PEGIEE (amino acids 735-740 inMyers et al (supra)) i.e., the 29th amino acid of the intracytoplasmicdomain. The plasmid in which the LAI sequence was inserted betweenvaccinia virus thymidine kinase (TK) gene homologous regions, was cutwith SphI and SmaI and further ligated with the MN gp120 sequence.VVTG9150 was then constructed b) conventional homologous recombinationand propagated for MN/LAI gp160 expression according to the conventionalmethod used for vCP205 on BHK21 cells. The protein was purified byimmunoaffinity chromatography.

1C—Experimental Procedure

Guinea pigs were submitted to immunization protocols as described inTable 1 hereinafter.

TABLE 1 Group # Inoculation days (Guinea-pig) D1 D29 1  4 μg gp160  4 μggp160 (1, 2, 3, 4, 5) 2 40 μg gp160 40 μg gp160 (6, 7, 8, 9, 10) 310^(6.1)  4 μg gp160 (11, 12, 13, 14, 15) CCID50 ALVAC-HIV 4 10^(6.1) 40μg gp160 (16, 17, 18, 19, 20) CCID50 ALVAC-HIV 5 10^(6.1) CCID50 ALVAC-10^(6.1) CCID50 ALVAC- (21, 22, 23, 24, 25) HIV + 4 μg gp160 HIV + 4 μggp160 6 10^(6.1) CCID50 ALVAC- 10^(6.1) CCID50 ALVAC- (26, 27, 28, 29,30) HIV + 40 μg gp160 HIV + 40 μg gp160 7 10^(6.1) CCID50 ALVAC-10^(6.1) CCID50 ALVAC- (31, 32, 33, 34, 35) HIV HIV

each dose was administered intramuscularly under a final volume of 1.2ml (0.6 ml in each thigh). When vCP205 and gp160 were both administered,these two products were mixed together before.

Serological analyses were carried out with blood samples collected ondays 0 (one day before the first immunization), 28. 43 and 57.Antibodies to HIV gp160 glycoprotein and V3 peptide titrated by ELISA asfollows:

Maxisorp F96 NUNC plates were coated for 1 hour at 37° C., thenovernight at 4° C. with one of the following antigens, diluted in 0.1 Mcarbonate buffer, pH 9.6:130 ng per well of purified gp160 MN/LAI; 200ng of V3 peptide from HIV MN.

Plates were then blocked for 1 hour at 37° C. with 150 μl of phosphatebuffered saline (PBS) pH 7.1-0.1% Tween 20-5% (w/v) powdered skim milk,(PBS-Tween-milk). All next incubations were carried out in a finalvolume of 100 μl, followed by 3 or 4 washings with PBS, pH 7.1-0.1%Tween 20.

Serial threefold dilutions of the sera, ranging from 1/100 to 1/24300 or1/1000 to 1/243000, in PBS-Tween-milk, were added to the wells andincubated for 90 min at 37° C. After washings (3 times), anti-guinea-pigIgG peroxydase conjugate (Sigma, rabbit IgG fraction) was diluted at1/3000 in PBS-Tween-milk, added to the plates and incubated for another90 min at 37° C. The plates were further washed (4 times) and incubatedin the dark for 30 min at room temperature with O-phenylenediaminedihydrochloride (Sigma) at 1.5 mg/ml in 0.05 M phosphate citrate buffer,pH 5.0 containing 0.03% sodium perborate (Sigma). The reactions werestopped with 50 μl of 4N H₂SO₄.

The optical density (OD) was measured at 490-650 nm with an automaticplate reader (Vmax, Molecular Devices). The blanks (mean value) weresubstracted to the data and duplicate values averaged. The antibodytiters were calculated for the OD value range of 0.2 to 1.3, from theregression curve of a standard hyperimmnune guinea-pig serum specificfor both gp160 and V3 antigens, present on each ELISA plate.

The titer of the standard serum had been previously determined accordingto the formula:${Titer} = {\log \quad {\frac{{OD}_{490 - 650} \times 10\quad \left( {{OD}\quad {value}\quad {range}\text{:}\quad 0.2\quad {to}\quad 1.3} \right)}{1/{dilution}}.}}$

ID—Serological Results

Averaged titers for each group of guinea pigs are presented in FIGS. 1(gp160 antibody titers) and 2 (V3 antibody titers).

Comparison of the anti-HIV antibody responses induced by gp160 alone(groups #1 and #2), vCP205 alone (group #7), and combination of bothantigens (groups #5 and #6)

Antibody Responses to gp160

The lowest responses were observed, after both the primary and boosterimmunizations, in guinea pigs that received 4 μg of gp160 (group #1).With 40 μg of gp160 (group #2), humoral responses were much moreelevated: only one inoculation was required for all animals toseroconvert, versus two with the 4 μg dose; and the mean antibody titersto V3 and gp160 were higher in group #2 than in group #1 (>+1 log higheron week 6).

vCP205 (10^(6.1) CCID50) injected alone (group #7) elicited anti-HIVantibodies at comparable but lower levels than those induced by gp160alone at 40 μg, especially after the booster injection (difference inmean titers ≈−0.4 log on week 6).

Mixing vCP205 with 4 μg of gp160 (group #5) was not found tosignificantly enhance the antibody response comparatively to vCP205alone. Conversely, and of great interest, two immunizations with thecombination vCP205 plus gp160 at 40 μg (group #6) induced the bestantibody titers, higher than those raised by vCP205 alone (group #7)(raise of mean ELISA titers ≈+0.8 log on week 6) and, in lesser extent,by 40 μg of gp160 alone (group #2)(≈0.4 log on week 6).

Antibody Responses to V3

Although the antibody titers raised against the V3 domain were, aspreviously observed, lower than those induced against whole gp160, thereactivity pattern to V3 was similar to that obtained to gp160. Inparticular, the (vCP205 plus 40 μg of gp160) combination proved to bethe best immunogen, whereas the 4 μg dose of gp160 injected alone wasthe worst.

Comparison of the anti-HIV antibody responses induced by the mixture ofvCP205 plus gp160 (groups #5 and #6) and by a prime (vCP205)/boost(gp160) immunization regimen (groups #3 and #4).

As observed in previous tests, a clear priming effect of vCP205 on theanti-HIV humoral responses following a boost with gp160 (at either 4 or40 μg) was found. Nonetheless, animals immunized according to thisprime/boost regimen displayed lower responses to V3 than thoseinoculated with two injections of the mixture vCP205 plus gp160 (using 4or 40 μg of gp160). Similar differences were seen when anti-gp160responses were considered, but only with 40 μg of gp160.

Noticeably, the prime/boost immunization using: (i) 40 μg of gp160(group #4) gave antibody levels equivalent to those elicited by twoinoculations of gp160 alone at 40 μg (group #2); or (ii) 4 μg of GP160(group #3) raised antibody titers similar to or lower than those inducedby two injections of vCP205 (group #7).

General Conclusion

Immunogenicity of the different combinations of ALVAC-HIV vCP205 and/orgp160 MN/LAI evaluated in the present study in guinea pigs can beclassified as followed:

gp160 (4 μg)<prime vCP205/boost gp160 (4 μg)=vCP205 vCP205+gp160 (4μg=prime vCP205/boost gp160 (40 μg) gp160 (40 μg)<vCP205+gp160 (40 μg).

In particular, these results revealed that two co-injections of vCP205and gp160 can induce higher anti-HIV serological responses (to V3 andgp160) than two inoculations of either vCP205 or gp160 alone, or than aprime (vCP205)/boost (gp160) immunization. Such an enhancing effect wasobserved mainly when vCP205 was combined with a high dose of gp160 (40μg) but not with a lower one (4 μg).

Example 2 Analysis of the Enhancing Effect of a Mixture vCP205+pg160MN/LAI on the Antibody Response to gp160 MN/LAI in Guinea-pigs

The experiment reported in Example 2 were performed in guinea-pigs (i)to confirm the ability of the mixture gp160 MN/LAI plus vCP205 tostimulate the antibody response to gp160, as previously observed inExample 1; (ii) to determine whether this enhancement results from asimple additive or rather a synergistic effect between the twoimmunogens; and (iii) to evaluate whether such an effect can be obtainedwhen the two products are inoculated simultaneously at distinct sites oronly when they are mixed.

2A—vCP205 Preparation was Achieved as Described in Example 1AHereinabove

2B—gp160 Preparation was Achieved as Described in Example 1B Hereinabove

2C—Experimental Procedures

Thirty-nine guinea pigs distributed in eight groups received vCP205and/or gp160 doses as stated in Table 2.

TABLE 2 ALVAC-HIV (vCP205) (CCID50) 0 10^(4.8) 10^(5.8) 10^(6.1) gp160 0# 11 to 14 # 21 to 24 # 36 to 40 (μg) 40 # 1 to 5  mixed mixedseparately # 15 to 19 # 25 to 30 # 31 to 35 80 # 6 to 10

Each guinea pig received intramuscularly two identical injections (eachunder a volume of 1.2 ml), one month apart. The viral vector and themixtures were administered in both thighs, whereas gp160 alone wasadministered in the right fore leg.

Serological analyses were carried out with blood samples collected ondays 1, 15, 28, 43 and 57. Antibodies to HIV gp160 MN/LAI glycoproteinand to non-recombinant purified canary pox (CPpp) were titrated by ELISAas described in Example 1C. To this end, 500 ng of CPpp/well were usedas well as a standard hyperimmune guinea-pig serum for CPpp.

2D—Serological Results

Anti-CPpp Antibody Response

The antibody response elicited against CPpp was measured in the threegroups of guinea pigs inoculated with 10^(4.8), 10^(5.8) or 10^(6.1)CCID50 of ALVAC-HIV (vCP205) alone (groups #3, #5 and #8, respectively).The mean titers of each group are presented in FIG. 3.

The doses of 10^(4.8) and 10^(5.8) CCID50 of vCP205 raised similaranti-CPpp antibody levels, which proved to be lower than those inducedby the dose of 10^(6.1) CCID50 of ALVAC-HIV, mostly after the firstinjection (difference in mean titers of ˜−0.7 log on week 4).

Anti-gp160 MN/LA! Antibody Response

The antibody response to gp160 MN/LAI was measured in all immunizedanimals. The mean titers of each group are represented in FIGS. 4a and 4b.

When the groups of guinea pigs were globally compared by varianceanalysis, a significant difference between immunogens was observed inthe antibody response elicited against gp160 (p<0.0005).

Injections of either gp160 MN/LAI at 40 or 80 μg (groups #1 and #2) orALVAC-HIV (vCP205) at 10^(5.8) or 10^(6.1) CCID50 (groups #5 and #8)were found to induce close anti-gp160 antibody levels which proved to bestatistically identical along the study. ALVAC-HIV (vCP205) at the doseof 10^(4.8) CCID50 (group #3) appeared to raise lower antibodyresponses, the difference in mean titers with groups #1, #2, #5 and #8ranging from −0.4 to 1.8 log during the serology, but statisticalsignificance was evidenced only with group #8.

These results suggested that the gp160-specific humoral responseelicited by the HIV protein at 40 to 80 μg or the recombinant ALVAC-HIV(vCP205) at 10^(5.8) or10^(6.1) CCID50 had reached its maximum. However,mixture 10^(4.8) CCID50 vCP205 plus 40 μg gp160 (group #4) was found toinduce elevated antibody titers which proved to be significantly higherthan those raised (i) by vCP205 alone at 10^(4.8), 10^(5.8) or 10^(6.1)CCID50 (difference in mean titers ranging from +0.5 to +2.6 log), and(ii) by gp160 alone at 40 or 80 μg (difference in mean titers rangingfrom +0.8 to +2.5 log).

The anti-gp160 antibody levels induced by the mixture vCP205 at 10^(5.8)CCID50 plus 40 μg gp160 (group #6) also appeared to be high and did notsignificantly differ from those elicited in group #4 (mixture withvCP205 at 10^(4.8) CCID50). Moreover, the simultaneous injection of10^(5.8) CCID50 vCP205 and 40 μg gp160 either mixed (group #6) orinjected separately (group #7) gave similar increased antibodyresponses, as confirmed statistically.

Whether or not the strongest anti-gp160 antibody responses observed withthe three combinations of vCP205 and gp160 (groups #4, #6 and #7)resulted from a simple additive or rather a synergistic effect betweenboth immunogens was difficult to assess. In an attempt to address thisissue, the mean ELISA titers measured experimentally for eachcombination were compared to the estimated titers that would result froman additive effect between gp160 and vCP205. As shown in Table 4, thetiters measured for the mixture with vCP205 at 10^(4.8) CCID50 (group#4) were found to be higher than the theoretical additive titers, theratio “measured titer/theoretical additive titer” ranging from 5.4 to165.5 along the serology. This ratio was also above 1 albeit neverexceeding 10, for the group receiving the mixture with vCP205 at10^(5.8) CCID50 (group #6). This was also true when gp160 wasadministered separately to vCP205 at the same dose (group #7), but onlyafter the primo immunization (weeks 2 and 4).

These results suggested that a synergism between ALVAC-HIV (vCP205) andgp160, potentiating the antibody response to gp160, can occur. Such aneffect would also take place when both immunogens are injectedseparately, although apparently less efficiently.

General Conclusion

The ability of the combination of gp160 MN/LAI (40 μg) and ALVAC-HIV(vCP205) (10^(4.8) or 10^(5.8) CCID50) to stimulate the humoral responseto gp160 MN/LAI in guinea pigs was confirmed. The antibody levelselicited against the HIV protein by these mixtures were indeed increasedcomparatively to those obtained by each immunogen at either a similar ora two-fold (or more) higher dose (i.e. gp160 at 40 or 80 μg or ALVAC-HIVat 10^(4.8), 10^(5.8) or 10^(6.1) CCID50).

This stimulating effect seemed to result from a synergistic rather thanan additive phenomenon, and could also occur at distance when bothantigens were injected at distinct sites.

Example 3 Comparison of the Immune Response Induced in Rhesus MacaguesEither by a Mixture of vCP205+gp160 MN/LAI or a Prime Boost ImmunizationvCP205/gp160 MN/LAI in Aluminum Hydroxide A1 (OH)₃(Alum)

3A—vCP205 Preparation was Achieved as Described in Example 1AHereinabove

3B—gp160 Preparation was Achieved as Described in Example 1B Hereinabove

3C—Experimental Procedure

Thirteen rhesus macaques (Macaca mulatta) were immunized according tothe immunization protocols as shown in Table 3.

TABLE 3 Immunizations (Weeks) Ma- Sex caques and Group num- # ber W0 W4W8 W12 W24 1 F1, gp160 gp160 gp160 gp160 gp160 F2 2 F4, gp160 + gp160 +gp160 + gp160 + gp160 + F5, alum alum alum alum alum M6 3 M11, AL-ALVAC- ALVAC- ALVAC- ALVAC- F12, VAC- HIV + HIV + HIV + HIV + M13, HIV +gp160 gp160 gp160 gp160 F18 gp160 4 F19, AL- ALVAC- gp160 + gp160 +gp160 + F20, VAC- HIV alum alum alum F21, HIV M22 F: female; M: male.

Macaques were administered doses intramuscularly in one thigh (right orleft alternatively), under a final volume of 1 ml, comprising 10^(6.5)CCID50 vCP205, 100 μg gp160 and/or 0.3 mg alum.

Blood samples were collected every two weeks, starting on week 0 (firstimmunization week).

Antibodies to HIV gp160 MN/LAI glycoprotein, V3 MN peptide, p24 LAI andCPpp were titrated by ELISA (FIGS. 5 to 8) as described in Example 1C.Reagent dosages were as follows: gp160 MN/LAI:130 ng/well; V3 MNpeptide:200 ng/well; p24 LAI: 130 ng/well; and CPpp:500 ng/well.

Two different peroxydase conjugates were used, diluted inPBS-Tween-milk, depending on the coating antigen:

for the gp160 MN/LAI, V3 MN and p24 LAI titrations: goat anti-monkey IgGperoxydase conjugate (Cappel, ref. 55432) at 1/1,000

for the CPpp titrations: sheep anti-human Ig peroxydase conjugate(Amersham, ref. NA 933) at 1/300.

Antibody titers were calculated for the OD value range of 0.2 to 1.3,from the regression curve of a standard specific hyperimmune macaqueserum present on each ELISA plate.

Neutralizing test were also carried out (FIG. 9). The assay determinesthe dilution of serum that prevents the development of syncytia in 50%of microwells infected with 10 CCID50 of HIV MN. The MN strain wasobtained from F. Barré-Sinoussi and propagated in CEM clone 166 cells.

Sera were decomplemented and twofold serial dilutions in RPMI beginning1/10 were prepared. Equal volumes of serum dilution and HIV suspension(500 μl each) were mixed and incubated for 2 hrs at 37° C. The HIVsuspension had been adjusted to contain 10² to 10^(2.5) CCID₅₀ per ml.

Prior to use, indicator CEMss cells were plated in microwells coatedwith poly-L-lysine, and incubated for 1 hr at 37° C. Culture medium wasremoved and replaced with the virus/serum mixtures (100 μl/well, 6 wellsper dilution). After 1 hr incubation at 37° C., culture medium was addedto each well and the plates were incubated at 37° C. All incubationswere done in a 5% CO₂ incubator.

After 7 and 14 days respectively, the cultures were examined under themicroscope and wells showing syncytia were recorded. Neutralizing 50%titer was computed according to SPEARMAN and KÄTER and expressed as thelog₁₀ of the end-point. As a confirmation, supernatants of the cultureswere collected on day seven, pooled for each dilution and assayed forreverse transcriptase (RT) activity.

Each assay included a set of uninfected microwells as negative controls,an infectivity titration of the virus suspension and a titration ofantibody in a reference serunm.

3D—Serological Results

The mean antibody kinetics are presented in FIGS. 5 to 9.

gp160 MN/LAI Antibodies

All animals injected with gp160 MN/LAI only (group #1) seroconverted,although weakly, to the HIV protein after one immunization andconsistently increased their response after the second and thirdinoculations (mean titers raised by +0.8 to +1.0 log two weekspost-injection). After the fourth immunization, titers reached similarlevels than after the third one, and then decreased. The lastinoculation induced a strong booster effect (mean titers raised by +1.3log two weeks post-injection) and elicited the highest titers of theperiod examined (5.0 log on week 26).

A marked adjuvant effect of alum (group #2) was observed on theanti-gp160 antibody response in naive macaques. Indeed, as compared tothe non-adjuvanted group (#1), the mean ELISA titers were enhanced by+1.0 to +2.0 log after each of the four first inoculations, and to alesser extent after the fifth injection (+0.3 to +0.5 log). The highestlevels of gp160-specific antibodies were obtained earlier than in group#1. This adjuvant effect was found to be significant (statisticalanalysis performed when possible, i.e. on weeks 4, 6 and 8, using theDunnett's t-test).

Interestingly, the mixture (ALVAC-HIV+gp160) (group #3) was found toinduce a significant higher response to gp160 than ALVAC-HIV after oneor two inoculation(s) (group #4) (difference in mean titers up to +1.5log). The anti-gp160 antibody titers were also more elevated in macaquesinjected with the mixture than in the vCP205-primed animals boosted withgp160 in alum (group #4). However, the differences were slight (+0.7 logmaximum) and found to be significant only on weeks 20, 24 and 28 (group#4) (Newman-Keuls test).

The combination (ALVAC-HIV+gp160) also proved to be a better immunogenthan gp160 alone (group #1) (mean titers between +0.8 to +1.7 log higheralong the experiment), and did not strongly differ from gp160 adjuvantedin alum (group #2) (differences in mean titers=+/−0.5 log).

Finally, the prime/boost immunization regimen (group #4) induced in mostcases higher antibody titers than inoculation with gp160 alone (group#1), especially after the gp160 boosts (differences up to +1.4 log), butlower responses than injection with gp160 in alum (group 42),particularly after the ALVAC priming (differences up to −2.0 log).

V3 MN Antibodies

On the whole, antibody responses elicited against V3MN shew a similarpattern than against gp160 MN/LAI, although to a lesser magnitude.

Alum (group #2) also increased the antibody titers to V3MN as comparedto the non-adjuvanted group (#1), and this enhancing effect was found tobe significant at weeks #2,4,6,8.

Animals injected with the mixture (ALVAC-HIV+gp160) (group #3) displayedsignificantly increased anti-V3MN responses than those receiving theprime/boost immunization (group #4) but only after the first and thesecond priming with ALVAC-HIV (weeks 4, 6 and 8) and following the lastgp160 boost (weeks 26 and 28) (Newman-Keuls test). Moreover, similarlyto what was seen on gp160, and although no statistical analysis could beperformed given the low number of animals tested, the mixture raisedV3MN responses higher than did gp160 alone (group #1) (titers augmentedby +1.0 to +1.8 log), and close to those induced by gp160 adjuvanted inalum (group #2) (titers=+/−0.5 log in most cases).

p24 LAI Antibodies

In the group of macaques injected with the mixture (ALVAC-HIV+gp160)(#4), 2 animals out of 4 developed an antibody response against p24 LAIas compared to the preimmune samples: #11 became positive after twoinoculations and titers increased by up to +1.3 log following the nextimmunizations; #18 clearly seroconverted after the third injection andmaintained or decreased its response afterwards.

In group #5 receiving the prime/boost immunization, only 1 or possibly 2from group #5 was (were) found to be positive on p24 LAI: #19 raisedantibodies as soon as the first ALVAC priming; #22 was hardly positiveafter the last gp160 boost.

Anti-canarypox (CPpp) Antibodies

All macaques immunized against ALVAC-HIV vCP205 either two (group #4) orfive (group #3) times elicited CPpp-specific antibodies two weeks afterthe first injection and reached their maximal responses after the secondinoculation (week 6). Following the gp160 boosts in group #4, theanti-CPpp titers gradually decreased and were reduced by—1.0 log on week28. In group #3, the mean antibody levels were maintained until week 14(two weeks after the fourth injection), diminished (−0.7 log), and thenincreased to their maximum after the last booster immunization (week26).

3E—HIV-1 MN Neutralizing Antibody Response

The mean titers of each group of macaques are presented in FIG. 9.

All the tested animals developed anti-HIV-1 MN neutralizing antibodieswhen examined after the fourth (week 16) and the fifth (week 26)injection, as compared to the preimmune samples (week 0).

Because of the low number of macaques studied in groups #1 and #2, nostatistical comparison could be performed for these animals. However,the lowest neutralizing titers were observed in group #1 inoculated withnon-adjuvanted gp160. In group #2 (except for week 26), injected withgp160 adjuvanted in alum, the neutralizing response was stronger than ingroup #1, similar on week 16 and higher on week 26 than in group #4(prime/boost immunization), and slightly lower than in group #3 injectedwith the (ALVAC-HIV+gp160) mixture.

Paired comparisons of groups #3 and #4 by the Newman-Keuls test revealedno statistical difference on week 16 but showed that the mixture(ALVAC-HIV+gp160) (group #3) induced significantly higher neutralizingtiters than the prime/boost immunization (group #4) on week 26.

General Conclusion

The present assay showed that the mixture vCP205 (10^(6.5) CCID₅₀) plusgp160 (100 μg) elicited significantly higher gp160 and V3-specificresponses than vCP205 or gp160 alone, and in some cases than theprime/boost immunization (vCP205/gp160 in alum), mainly after the finalgp160 booster injection. The vCP205+gp16 mixture proved to be similarlyimmunogenic to gp160 adjuvanted in alum; given the low number of animalsstudied in the other groups. Moreover, the mixture appeared to evoke thebest seroneutralizing responses to HIV-1-MN after the last fifthinjection, although significance of this result could be proven onlywhen compared with the prime/boost immunization, given the low number ofanimals in the other groups.

Example 4 Immunogenicity of Purified gp160 MN/LAI in the Absence orPresence of Canarypox (ALVAC), in Guinea-pigs

The experiment reported in the present Example 4 shows that both crudeand purified non-recombinant ALVAC (CPpp) display adjuvant properties.

4A—CPpp Preparations

CPpp (ALVAC) is derived from a canarypox strain isolated from a poxlesion on a infected canary, as described in Tartaglia et al, Virology(1992) 188:217. CPpp is produced on chick embryo fibroblasts in DMEM-HamF12, washed without serum and resuspended in lactoglutamate (crudeCPpp). Instead of being resuspended in lactoglutamate, purified CPpp isobtained according to the purification process described in Joklick etal, Virology (1962) 18:9.

4B—gp160 MN/LAI Preparation

gp160 preparations were achieved as descibed in example 1B

4C—Experimental Procedure

Guinea pigs were submitted to immunization protocols as described inTable 4 hereinafter.

TABLE 4 Primo-immunization Booster (D1) (D29) Group gp160 ALVAC ALVACdose gp160 (Guinea-pig #) dose (μg) (CPpp) (CCID50) dose (μg) 1 5 None 05 (1, 2, 3, 4, 5) 2 5 Crude 10⁶ 5 (6, 7, 8, 9, 10) 3 5 10⁶ 5 (11, 12,13, 14, 15) 4 5 Purified 10⁷ 5 (16, 17, 18, 19, 20) 5 5 10⁸ 5 (21, 22,23, 24, 25)

Animals received both the primo and booster 1.10 ml dosesintramuscularly (0.55 ml in each thigh) one month apart.

Serological analyses were carried out as described in Example 1C, usingblood samples collected at days −1, 28 and 56.

4D—Serological Analyses

Serological analyses were carried out with blood samples collected ondays −1. (one day before the first immunization), 28, and 56. Antibodiesto HIV gp160 glycoprotein and CPpp were titrated by ELISA using the sameprocedure as described in example 1C

4E—Serological Results

Anti-CPpp Antibodies (FIG. 10a)

Four weeks after the first immunization, all the animals seroconverted(except group #1 which did not received any CPpp), and the titersremained stable after the gp160 booster till week 8.

Response to canarypox induced by 10⁶ CCID₅₀ of crude CPpp wassignificantly higher (+0.7 to 0.8 logs) than the one raised with thesame dose of purified virus, was comparable to that elicited by 10⁷CCID₅₀ of purified CPpp, and was lower (˜−0.8 log) than that obtainedwith the dose of 10⁸ CCID₅₀ of purified CPpp.

Anti-HIV gp160 MN/LAI Antibodies (FIG. 10b)

Anti-gp160 MN/LAI antibodies were elicited during the four weeksfollowing the first injection in all animals, except some in group #5.In this group, which received a mixture of gp160 and 10⁸ CCID₅₀ ofpurified CPpp, only 3 animals out of 5 seroconverted to gp160. For eachguinea pig, a booster effect was noticeable after the second injectionof 5 μg of gp160.

The best anti-gp160 antibody responses were obtained in group #3, primedwith gp160 mixed with the lowest dose (10⁶ CCID₅₀) of purified CPpp.Indeed, this group displayed a significant increase in antibody titers(+0.8 and +0.9 logs at weeks 4 and 8, respectively), comparatively togroup #1 inoculated with the protein alone.

Co-injection of 10⁷ CCID₅₀ of purified CPpp with gp160 (group #4) alsoenhanced the humoral response as compared to injection of the proteinalone, but only on week 8 after the gp160 boost (+0.7 log).Surprisingly, in group #5 (gp160 mixed with 10⁸ CCID₅₀ of purifiedCPpp), a significant decrease in responding animals was observed (3 outof 5, versus 5 out of 5 in all other tested conditions). Moreover, themean antibody titer (2.352 log) of the positive guinea pigs from group#5 was the lowest obtained in this assay.

Nevertheless, such a CPpp-induced inhibitory effect did not have anyinfluence on the secondary response to gp160, which reached similarlevels to those obtained in group #1.

Noticeably, addition of 10⁶ CCID₅₀ of crude CPpp to gp160 did notimprove the antibody response as compared to gp160 alone.

General Conclusion

This study clearly demonstrates an adjuvant effect of crude and purifiedCPpp on the immunogenicity of gp160 MN/LAI inoculated IM in guinea pigs.Such a stimulation of the anti-gp160 antibody response was mostlyobserved at 10⁶ CCID₅₀ of purified CPpp, whereas a marked inhibitoryeffect was noted at the higher dose of 10⁸ CCID₅₀.

The results obtained with crude CPpp at 10⁶ CCID₅₀ indicates that thisCPpp preparation does not seem to be able to enhance the anti-gp160humoral response when combined with the 5 μg dose of the tested gp160.However, the same preparation does enhance the response to 1 μg gp160(data not shown). Accordingly, the crude CPpp immunomodulating effectseems to be gp160-dose dependent.

Altogether, these findings show that both CPpp and gp160 must be used atoptimal concentrations to see an adjuvant effect of canarypox. Thepresent observation that both crude and purified CPpp can stimulate theanti-gp160 antibody response is in favor of the hypothesis that CPpp hasintrinsic immuno-stimulating properties.

Example 5 Immunogenicity of gp160 MN/LAI in the Presence of PurifiedALVAC-Luc (vCP292) Inactivated or not, in Guinea-pigs

5A—vCP297 Preparation

vCP297 is an ALVAC vector derived from CPpp by homologous recombinationso as to produce a vector in which the luciferase encoding sequence isplaced under the control of an ALVAC promoter. vCP297 is produced andpurified as described in Example 4A.

One ml of a vCP297 preparation exhibiting a mean titer of 10^(9.3)CCID₅₀ on QT35 cells, was diluted 1/10 in PBS without Cask and Mg⁺⁺ andinactivated at 56° C., 7 hours. It was then centrifuged during 5 hoursat 10.000 rpm (centrifuge Sigman 201 M) and the pellet and supernatantwere harvested separately. The protein quantity and residual viral titerwere quantified, being respectively 55 μg/ml and 10^(3.5) CCID50/ml forthe pellet and ≈1 μg/ml and 10^(0.3) CCID50/ml for the supernatant.

5B—gp160 Preparations Were Achieved as Described in Example 1B.

5C—Experimental Procedure

Guinea pigs were submitted to immunization protocols as described inTable 5 hereinafter.

TABLE 5 First immunization (D1) Purified ALVAC- Luc (vCP297) Boostergp160 Infectious (D29) Group MN/LAI Proteins dose gp160 MN/LAI(Guinea-pig #) doses (μg) (μg) (CCID50) doses (μg) 1 5 0 0 5 (1, 2, 3,4, 5) 2 0.055 10⁵ (6, 7, 8, 9, 10) 3 0.55 10⁶ (11, 12, 13, 14, 15) 4pelleted fraction of (16, 17, 18, 19, 20) the inactivated virus 0.55 10^(1.5) 5 supernatant of the (21, 22, 23, 24, 25) inactivated virusafter centrifugation¶ ˜1 =10^(0.3)

Animals received the primo and booster doses under a final volume 1.10ml, intramuscularly (0.55 ml in each thigh), one month apart.

Serological analyses were carried out as described in Example 1C, usingblood samples collected at days −1, 28 and 56.

The isotypic distribution of the anti-gp160 humoral response wasmeasured at day 56, using the procedure and conditions described inExamples 1C and 2C. The only modification was the use of distinctperoxydase-conjugated goat antibodies specific for guinea-pig isotypeIgG1 (Nordic, ref.: GAGp/IgG1/PO) or IgG2 (Nordic, ref.: GAGp/IgG2/PO),diluted 1/3.000 in PBS-Tween-milk.

5D—Serological Results

Anti-CPpp Antibodies (FIG. 11a)

As previously observed, the humoral response induced against CPpp wasdose-dependent: only 3 out of 5 guinea-pigs immunized with 10⁵ CCID₅₀ ofpurified ALVAC-Luc (vCP297) (group #2) weakly seroconverted to CPpp,whereas all animals (out of 5) that received 10⁶ CCID₅₀ of the purifiedvirus (group #3) developed a CPpp-specific response, and at much higherlevels (mean ELISA titer in group #3˜2 logs higher than in group #2).

The anti-CPpp titers elicited by the pelleted fraction of theinactivated ALVAC-Luc (group #4) were similar to those induced by thenon-inactivated virus at equivalent protein quantity (group #3).

Surprisingly, the supernatant of inactivation of vCP297 (group #5) wasalso able to mount an antibody response to the canarypox, and the titersinduced were the highest observed in this assay. In particular, such aresponse differed in average by +0.6 and +0.9 log, on week 4 and 8respectively, with that elicited by the non-inactivated purified virus(group #3). The high protein content present in thissupernatant—measured subsequently to inoculation—reaching ˜1 μg versus0.55 μg for both the non-inactivated virus (group #3) or the pelletedfraction of the inactivated virus (group #4) could account for suchresults.

Anti-gp160MN/LAI Antibodies (FIG. 11b)

Anti-gp160MN/LAI antibodies were elicited in all animals during the fourweeks following the first injection. For each guinea pig, an anarnnesticresponse was noticeable after the gp160 booster injection.

While no significant difference in anti-gp160 antibody titers wasdetected between the five groups of guinea pigs after theprimo-immunization, an enhancement of the humoral response to the HIVantigen was observed in some groups after the second inoculation.Indeed, by variance analysis using the Dunnett's t-test, thegp160-specific ELISA titers were found to be significantly higher ingroups #3 and #4 than in group #1 (mean titers on week 8 in both groups#3 and 4 raised by +0.7 log as compared to group #1). In other words,these findings indicated that purified ALVAC-Luc, either inactivated ornot, at protein quantity corresponding to 10⁶ CCID₅₀ of infectiousvirus, had a significant adjuvant effect on the anti-gp160 antibodysecondary response.

Priming with gp160 and purified ALVAC-Luc at 10⁵ CCID₅₀ (group #2) alsoincreased the anti-gp160 response (mean titers on week 8 raised by +0.4log as compared to group #1), but such a stimulation was not found to besignificant using the Dunnett's t-test.

By contrast, a significant adjuvant effect was detected in group #5,co-injected with gp160MN/LAI and the supernatant of inactivated purifiedALVAC-Luc, (mean titers on week 8 raised by +0.5 log as compared togroup #1), in accordance with the high protein content of ALVAC-Lucorigin found in the supernatant.

Noticeably, the stimulating effect on the anti-gp160 humoral responseassociated to ALVAC-Luc, or products derived from it, was not found tobe strictly related to the intensity of the anti-CPpp antibody responseelicited. This confirms previous observations in Example 1, showing thathigh anti-CPpp titers were inversely related to anti-gp160 antibodylevels, probably as a consequence of antigenic competition between theHIV glycoprotein and the high doses of ALVAC injected.

IgG1 and IgG2 Isotypic Profiles of the Anti-gp160 Antibody Response

The co-injection of gp160 and ALVAC-Luc, either inactivated or not (atprotein quantity corresponding to 10⁶ CCID₅₀ of infectious virus), wasfound to significantly increase the anti-gp160 antibody response of theIgG2 isotype, but not of the IgG1 one. Such an elevated IgG2 responsewas detected neither in group #2, that received gp160MN/LAI and 10⁵CCID₅₀ of purified recombinant canarypox, nor in group #5, injected withgp160MN/LAI and the supernatant of inactivated ALVAC-Luc.

General Conclusion

The data presented herein confirm those obtained in Example 1 withpurified CPpp, showing that purified recombinant canarypox ALVAC-Luc(vCP297), when co-injected with gp160MN/LAI at the dose of 10⁶ CCID₅₀ inguinea-pig, had also the capacity to significantly: (1) stimulate thegp160-specific IgG secondary response; and (2) influence the isotypicprofile of the anti-gp160 antibodies (increase in specific IgG2 titers).However, this adjuvant effect was detected earlier with CPpp than withvCP297 (i.e., after the primo-immunization for the former versus onlythe gp160 boost for the latter), suggesting that recombinant ALVAC-Lucmight be less effective in enhancing the humoral response than theparental vector.

Infectivity of ALVAC-Luc was not required for such a stimulating effectto occur, since both the non-inactivated and heat-inactivatedrecombinant canarypox, at equivalent protein quantity (corresponding tothat contained in 10⁶ CCID₅₀ of infectious virus), induced similarenhanced anti-gp160 antibody titers.

The observation that the supernatant of inactivated purified ALVAC-Lucalso displayed an adjuvant effect on the anti-gp160 antibody responsewas unexpected, but could be explained by its high protein content ofALVAC-Luc origin. Its ability to elicit the highest antibody titersagainst CPpp but not against gp160, confirms the results obtained inExample 1 using various doses of purified ALVAC.

Altogether, these findings are in line with the previous hypothesis thatthe canarypoxvirus induces some immunomodulating effects in vivo.

Example 6 Immunogenicity and Efficacy of a Detergent-splitted MonovalentA/Texas Flu Vaccine in the Absence or Presence of Canarypox (ALVAC) inMice

The experiment reported in the present example 6 shows thatnon-recombinant ALVAC increases the immunogenicity and the efficacy of adetergent-splitted flu vaccine essentially in aged immunocompromisedmice.

6A—CPpp Preparation

CPpp preparations were achieved as described in Example 4A. The titer ofthe stock CPpp preparation is 1.6 10⁹CCID 50/ml.

6B—Detergent-splitted Monovalent A/Texas Flu Vaccine Preparation

The detergent-splitted monovalent A/Texas flu vaccine (A/Texas) wasmanufactured by Connaught laboratories and dialyzed against PBS beforeuse, to eliminate residual detergent and formol from the vaccine.

6C—Serological Analyses

Serological analyses were cared out with blood samples collected on days−4 (4 days before the first immunization), 14 and 35. Antibodies to HAwere titrated as follows: Wells of Maxisorp F96 NUNC plates were coatedwith 1 μg/ml of HA in a Carbonate buffer 0.1 M, pH 9.6 overnight at roomtemperature. Plates were then blocked for 1 hour at room temperatureWith 200 μl of 0.1% BSA (Bovine Serum Albumin) in PBS Followed by 4washings in washing buffer (PBS/0.1% Tween 20). All next incubationswere carried out in a final volume of 100 μl, followed by 5 washings inwashing buffer.

Serial threefold dilutions of sera in dilution buffer (PBS/0.1% Tween20/0.1% BSA) ranging from 1/100 to 1/218700 were added to the wells andincubated 60 min at 37° C. After washings, a Sheep anti-mouse IgG1peroxydase conjugated (Serotec) 1/15000 diluted or a Goat anti-mouseIgG2a Horseradish peroxydase conjugated (Caltag laboratories)1/30000diluted were added to the plates and incubated for another 60 min at 37°C. The plates were farther washed and incubated for 20 min withO-phenylenediamine dihydrochloride (Sigma) at 1.5 mg/ml in 0.05 Mphosphate citrate buffer, pH 5.0 containing 0.03% sodium perborate(Sigma). The colored reactions were stopped with 50 μl of 4N H₂SO₄.Absorbance was read in a Titer Multiscan plate reader at 450 nm. Theantibody titers were measured as the reciprocal of the last dilution atwhich the absorbance was 2 fold over the background absorbance obtainedwith pre-immune sera.

6D—Challenge

Randomized groups of mice were challenged on day 42 with 50 μl of livemouse-adapted A/Taiwan/1/86 influenza virus (H1 strain) corresponding to5 lethal doses 50 of virus (5 LD₅₀). The infectious doses were givenintranasally after slight anesthesia of mice with Isoflurane. Theprotective immune responses induced by the tested vaccinal compositionswere assessed by means of survival yields and weight changes that is agood parameter of morbidity. Mortality and weight changes in the micewere monitored daily and every pair day respectively up to 21 days afterchallenge. The article Suryaprakash S and al, (1997), 96:157-169 iscited by reference for achieving experimental challenges.

6E—Immunization

Six randomized groups of 16-to-18 month old (aged) or 2-month old(young) DBA/2 mice were each submitted to one of the immunizationprotocol as described in Table 6 hereinafter. Each group is constitutedwith 6 mice.

TABLE 6 Primo-immunization Boost A/Texas ALVAC (CPpp) ALVAC (CPpp) Groupdose dose A/Texas dose dose DBA/2 (in μg) (in CCID 50) (in μg) (in CCID50) 1 3 0 3 0 2 3 2 × 10⁷ 3 2 × 10⁷ 3 0 2 × 10⁷ 0 2 × 10⁷

The groups were primed and boosted, via the S.C. route, with thecompositions in a final volume of 0.2 ml. For immunization of group 2,A/Texas and appropriate amount of ALVAC were mixed together withappropriate amount of PBS to bring the final injected volume to 0.2 mlper mouse. The booster immunization was carried out in all groups onemonth later.

6F—Serological Results

Anti A/Texas IgG1 Antibodies (FIG. 12)

Anti-A/Texas IgG1 antibodies were elicited during the two weeksfollowing the first injection in 3 to 6 mice from group 1, in 5 to 6mice from group 2, whereas no specific IgG1 were elicited in mice primedwith ALVAC alone (group 3). The specific IgG1 mean titer wasapproximately 10 fold higher in the group of mice primed with themixture of A/Texas and CPpp (group 1) than that observed in the group ofmice given A/Texas alone (group 2). The boost did not change thedistribution pattern of specific IgG1 responses (observed in the 3groups of mice) during the 15 days following the second injection.However, the specific IgG1 mean titers of groups 1 and 2 were ten-foldhigher.

Anti A/Texas IgG2a Antibodies (FIG. 13)

Anti-A/Texas IgG2a antibodies were elicited during the two weeksfollowing-the first injection in 3 to 6 mice from group 1, in 5 to 6mice from group 2, whereas no specific IgG2a were elicited in miceprimed with ALVAC alone (group 3). The specific IgG2a mean titer wasapproximately 10 fold higher in the group of mice primed with themixture of A/Texas and CPpp (group 1) than that observed in the group ofmice given A/Texas alone (group 2). The boost did not change thedistribution pattern of specific IgG2a responses (observed in the 3groups of mice) during the 15 days following the second injection.However, the specific IgG2a mean titers of groups 1 and 2 were ten-foldhigher.

General Conclusion

This study clearly demonstrates an adjuvant effect of CPpp on theimmunogenicity of A/Texas inoculated subcutaneously in immunocompromisedaged mice. A similar enhancer supportive effect of CPpp on theimmunogenicity of A/Texas is also observed in young mice. It is alsoworth noticing that CPpp increases both specific IgG2a and IgG1responses in old mice immunized with the mixture of ALVAC and A/Texas;which means that CPpp could act both on TH1 (T helper 1) and TH2 (Thelper 2) immune responses. Indeed, it is well understood for a manskilled in the art that the, TH2 immune response correlates with thelevel of specific IgG1 response in mice and is featured by a ratherhumoral immune response, whereas the TH1 immune response correlates withthe level of specific IgG2a response and is commonly featured by acytotoxic and inflammatory immune response. In conclusion, this revealsthat CPpp acts both on specific cellular and humoral immune responses,when it is concomitantly administered with an immunogenic compound.

6G—Challenge Results (FIGS. 14 and 15)

Mortality (FIG. 14)

Three weeks after the boost, all the aged mice were given intranasally alethal challenge of live influenza virus. All the 6 mice of the group 3(group receiving CPpp alone) died during the 8 days consecutive tochallenge. Only, 1 of 6 mice (16% survival rate) of the group 1 (groupreceiving A/Texas alone) was still alive 20 days after challenge whereas4 of 6 mice of the group 2(group receiving the mixture A/Texas and CPpp)(66% survival rate) were still alive. Moreover, the survival curve ofgroup 2 clearly shows that the two deaths observed were delayed comparedto those observed in groups 1 and 3 (FIG. 14)

Morbidity (FIG. 15)

The morbidity of mice after challenge was monitored for 20 days andassessed by the weight loss rate. The weight loss occurred shortly afterthe challenge in the group of mice immunized with CPpp alone (group 3)reaching up to 35% of the initial weight. Mice immunized with A/Texasalone (group 1) also showed a severe weight loss after challenge similarto that observed in group 3. The weight loss rate curve during the 20days of the monitoring for the only one survivor of group 1 isrepresented in FIG. 15 and clearly shows that the weight loss was fastand severe, whereas the weight recovery was much slower. On the otherhand, the weight loss rate curve involving the 4 survivors of groupimmunized with the mixture of ALVAC and A/Texas (group 2) showsimprovements over group 1. First, the maximum weight loss rate did notexceed 15% of the initial weight and second, the weight recovery wasfaster, since the survivors had recovered their initial weight by theend of the monitoring.

Although morbidity and mortality results about aged mice only arereported here, it is indicated that similar results were obtained withyoung mice.

General Conclusion

Morbidity and mortality results obtained with the live influenzachallenge model are in agreement with those obtained from immunogenicitystudies and show that ALVAC is not only able to enhance the specificimmune response to A/Texas but also contributes to the elicitation of aspecific protective immune response, when it is co-administered with anantigen from a pathogenic micro-organism.

What is claimed is:
 1. A composition comprising (i) an immunogeniccompound and (ii) a poxvirus encoding an heterologous polypeptide whichis selected from the group consisting of adhesion molecules,co-immunostimulatory molecules, chemokines apoptotic factors, cytokinesand growth hormones.
 2. The composition according to claim 1, whereinthe immunogenic compound is selected from the group consisting of apeptide, a polypeptide, a DNA plasmid encoding a peptide or apolypeptide, and an hapten coupled to a carrier molecule.
 3. Acomposition comprising (i) an immunogenic compound which is a firstpolypeptide, and (ii) a poxvirus encoding a second heterologouspolypeptide, which has an amino acid sequence identical to the aminoacid sequence of the first polypeptide.
 4. A composition comprising (i)an immunogenic compound which is a DNA plasmid encoding a firstpolypeptide, and (ii) a poxvirus encoding a second heterologouspolypeptide, which has an amino acid sequence identical to the aminoacid sequence of the first polypeptide.
 5. The composition according toclaim 2, wherein the first and second polypeptide are HIV or influenzavirus polypeptides.
 6. A method of enhancing a specific immune responseto an immunogenic compound in a vertebrate, the method comprisingadministering to the vertebrate a composition comprising a poxvirus andthe immunogenic compound.
 7. The method according to claim 6, whereinthe immunogenic compound comprises at least one antigenic determinant ofa pathogenic microorganism or a tumor-associated antigen.
 8. The methodaccording to claim 7, wherein a protective immune response against thepathogenic microorganism or tumor is induced.
 9. The method according toclaim 8, wherein said administering treats or prevents an infectiousdisease induced by the pathogenic microorganism or a disorder associatedwith the tumor.
 10. The method according to claim 6, wherein theimmunogenic compound is a peptide or a polypeptide.
 11. The methodaccording to claim 10, wherein the peptide or polypeptide is from apathogenic microorganism or is tumor associated antigen.
 12. The methodaccording to claim 10, wherein the peptide or polypeptide is from HIV oran influenza virus.
 13. The method according to claim 6, wherein theimmunogenic compound is a recombinant DNA plasmid encoding a peptide ora polypeptide that comprises at least one antigenic determinant of apathogenic microorganism or tumor-associate antigen.
 14. The methodaccording to claim 13, wherein the administering induces a protectiveimmune response against the pathogenic microorganism or tumor.
 15. Themethod according to claim 14, wherein said administering treats orprevents an infectious disease induced by the pathogenic microorganismor a disorder associated with the tumor.
 16. The method according toclaim 13, wherein the peptide or polypeptide is from HIV or influenzavirus.
 17. The method according to claim 6, wherein the immunogeniccompound is an hapten coupled to a carrier molecule.
 18. The methodaccording to claim 6, wherein the poxvirus is a live virus.
 19. Themethod according to claim 18, wherein the poxvirus is attenuated. 20.The method according to claim 6, wherein the poxvirus is an inactivatedvirus.
 21. The method according to claim 6, wherein the poxvirus doesnot encode a heterologous polypeptide.
 22. The method according to claim6, wherein the poxvirus encodes a heterologous polypeptide.
 23. Themethod according to claim 22, wherein the heterologous polypeptide isselected from the group consisting of adhesion molecules,co-immunostimulatory molecules, apoptotic factors, cytokines and growthhormones.
 24. The method according to claim 22, wherein the heterologouspolypeptide is different from and immunologically cross-reactive withthe immunogenic compound.
 25. The method according to claim 22, whereinthe heterologous polypeptide has an amino acid sequence identical to theimmunogenic compound.
 26. The method according to claim 6, wherein thepoxvirus is selected from the group consisting of orthopoxvirus,avipoxvirus, capriposvirus, suipoxvirus, molluscipoxvirus, yataposvirus,or an entomopoxvirus.
 27. The method according to claim 6 wherein thepoxvirus is a vaccinia virus.
 28. The method according to claim 6wherein the poxvirus is a canarypoxvirus.